Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein ␤ homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1.

Asparaginase reduces liver protein synthesis by increasing the phosphorylation of eukaryotic initiation factor 2 (p‐eIF2) and inhibiting signaling downstream of mTORC1. The objective of this study was to assess the relationship between the eIF2 kinase, GCN2, and mTOR in the hepatic response to the anti‐leukemic drug, asparaginase. GCN2+/+ and GCN2−/− mice (n=6 per group) were administered PBS or asparaginase (3 IU g/kg Elspar) 30 min after injection with either rapamycin (2 mg/kg) or PBS. Mice were killed and livers collected 8h later. Asparaginase, alone or in combination with rapamycin, significantly increased p‐eIF2 in the liver of GCN2+/+ but not GCN2−/− mice, whereas rapamycin alone reduced p‐eIF2 in GCN2+/+ mice only. In the livers of GCN2+/+ mice, the phosphorylation states of two markers of mTORC1 activity, namely p‐4E‐BP1 and p‐S6K1, were slightly reduced by asparaginase, moderately reduced by rapamycin, and maximally inhibited by the drug combination. In the livers of GCN2−/− mice, both p‐S6K1 and p‐4E‐BP1 were significantly reduced by rapamycin. On the other hand, p‐S6K1 and p‐4E‐BP1 were derepressed following asparaginase treatment, with values similar to PBS‐injected controls. These results demonstrate that asparaginase inhibits mTORC1 signaling in liver by a GCN2‐dependent and rapamycin‐independent pathway. Funded by AICR and IUSM